Originally published online as doi:10.1189/jlb.0103041 on June 16, 2003
Published online before print June 16, 2003
(Journal of Leukocyte Biology. 2003;74:151-160.)
© 2003
by Society for Leukocyte Biology
Retinal transplantation: progress and problems in clinical application
R. D. Lund*,
S. J. Ono
,
D. J. Keegan
and
J. M. Lawrence
* Moran Eye Center, University of Utah, Salt Lake City; and
Department of Ocular Immunology and
Transplantation Unit, Institute of Ophthalmology, University College, London, United Kingdom
Correspondence: Professor R. D. Lund, Moran Eye Center, North Medical Drive, University of Utah Health Science Center, Salt Lake City, UT 84132. E-mail: raymond.lund{at}hsc.utah.edu
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ABSTRACT
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There is currently no real treatment for blinding disorders that stem from the degeneration of cells in the retina and affect at least 50 million individuals worldwide. The excitement that accompanied the first studies showing the potential of retinal cell transplantation to alleviate the progress of blindness in such diseases as retinitis pigmentosa and age-related macular degeneration has lost some of its momentum, as attempts to apply research to the clinic have failed so far to provide effective treatments. What these studies have shown, however, is not that the approach is flawed but rather that the steps that need to be taken to achieve a viable, clinical treatment are many. This review summarizes the course of retinal transplant studies and points to obstacles that still need to be overcome to improve graft survival and efficacy and to develop a protocol that is effective in a clinical setting. Emphasis is given particularly to the consequences of introducing transplants to sites that have been considered immunologically privileged and to the role of the major histocompatibility complex classes I and II molecules in graft survival and rejection.
Key Words: photoreceptor degeneration • grafting • immune rejection
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INTRODUCTION
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This review considers two areas of transplantation involving retina. In the first, retinas were dissected from immature donors and implanted over the brain, mostly of young rats, to ask questions of developmental neural specificity and to assess the capacity of these grafts to relay visual information to the host brain to affect responses. The second involved the implantation of cells to the subretinal space [between the outer retinal layers and the retinal pigment epithelium (RPE)] of animals with retinal degenerative diseases, which ultimately lead to photoreceptor death. The object of this approach is to limit the progress of photoreceptor loss or to replace lost photoreceptors with new ones. For the two approaches, there are some common questions: Will the transplanted cells function normally in their ectopic location? Will they support visual function? Will they be tolerated by the host inflammatory systems? Will the host immune system react to their presence even though they have been placed in classically immune-privileged sites?
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INTRACEREBRAL RETINAL TRANSPLANTATION
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Retinal transplantation has a long history of application in nonmammalian vertebrates as a tool for studying issues of neural development and regeneration, including such problems as neural determination and neural recognition (e.g., refs. [1
2
3
]). This work was extended to mammals in a study by Tansley [4
], who was able to show that embryonic retina could survive implantation over the brain of neonatal rats. Subsequently, a series of studies using this preparation, which was directed primarily toward examining the specificity of neural connections formed by a developing retina [5
], was undertaken in rodents. These transplants were placed in an ectopic location, and the axons growing from them entered the central nervous system (CNS) by an anomalous route. It was found that ganglion cell axons from these ectopic retinas grew into the brain, often taking quite abnormal courses and using substrates very different from those of the normal, optic pathway to reach the optic targets in the brain. The new connections formed elicited physiological responses in postsynaptic cells [6
]. Furthermore, simple responses such as light avoidance were relayed through these transplants [7
, 8
], and a pupillary light reflex could be elicited in the host eye by photic stimulation of the transplant [9
]. More complex behavioral analysis showed that animals were "aware" of the visual input delivered by these grafts and that there were elaborate interactions between different functional tasks, as the salience of that input had to be first reinforced in a training paradigm [10
, 11
]. This body of work paralleled other studies regarding transplantation in rodent models of Parkinson’s disease (e.g., ref. [12
]) and later in the use of sciatic nerve grafts to promote regeneration of injured optic pathways including those from the eye to the brain (e.g., ref. [13
]). The Parkinson’s disease model was taken to patients with some success, although it is still true some 20 years after the first attempts at transplanting to patients that this is far from a routine therapy [14
, 15
].
One issue that was largely ignored in the early studies was the possibility of graft rejection. It was assumed that the brain was an immunologically privileged site and that immune suppression was unnecessary. Some of the laboratories had highly inbred animal colonies, and so donor-host disparities may not have been sufficiently large to initiate rejection; in our studies, we were using immature recipients for much of the work, and this also helped to reduce problems. However, as more work was done, it became clear that grafts were not always protected, and a literature developed showing the need for caution (e.g., refs. [16
17
18
]). Such studies illustrated the importance of understanding and defining what was meant by "immune privilege" in central neural tissues. The retinal graft studies showed that retinas taken from E12 embryos could survive grafting to neonatal hosts, although allogeneic donor/host matches varied on major histocompatibility complex (MHC) and minor histocompatibility complex antigens, and an unexpectedly high percentage of mouse-to-rat xenografts survived [19
, 20
] and could function [21
]. It should be noted that mismatched grafts placed in hosts older than 
10 days of age were susceptible to rejection. Rejection was also seen in other transplant paradigms involving embryonic donor and adult hosts, and the detailed course of this reaction was described in one of these [17
]. It became clear that the surviving retinal grafts, even to neonatal hosts, were not completely protected but could also be induced to undergo rejection under a range of different circumstances. First, if the blood/brain barrier (BBB) was opened transiently with mannitol, a massive infiltration of lymphocytes and evidence of graft destruction were seen [22
]. Second, if a skin graft matching the donor genotype was placed on the skin, graft rejection was induced. This was studied further using a set of rat strains that varied in specific MHC and minor histocompatibility antigens; the results showed that a major determinant of allogeneic, neural graft rejection was the disparity of MHC antigens. Disparity in non-MHC antigens resulted in only mild-to-moderate rejection responses [23
]. The progress of rejection could be monitored functionally, and this was done using the pupillary light reflex [24
]. It was found that although the first invasion of lymphocytes occurred 5–7 days after introducing a mismatched skin graft, there was an early functional enhancement of the pupillary response, which was accompanied by up-regulation of classes I and II MHC expression by microglia. These had invaded the graft from the host brain. Somewhat unexpectedly, we could also induce rejection of surviving grafts by inducing degeneration of host neural pathways in the vicinity of the graft or of its output axons [20
, 25
]. For example, if an embryonic mouse retina was implanted over the midbrain of a newborn rat, and it subsequently made neural connections with the midbrain optic center, the superior colliculus, later damage to the host optic nerve led to rejection of the retinal graft. Enigmatically, similar host optic pathway damage made at the time of transplantation did not lead to graft rejection. Finally, administration of interferon-
also led to graft rejection [26
].
A unifying theme in this body of work showed that each of the perturbations—opening the BBB, skin grafting, and CNS lesions—resulted in MHC expression being induced in microglia. With suitable donor/host combinations, the skin-grafting studies showed that the response was haplotype-specific and not a generalized response to nonspecific inflammatory reactions. In the case of neural degeneration, it was found that damage to neural pathways resulted in microglia, which previously had not expressed class I or class II MHC antigens, expressing both [27
]. This suggests that up-regulation of expression as a result of unrelated events can, by introducing an afferent limb to the immune response, precipitate a rejection reaction. It is interesting that microglia within degenerating, unmyelinated pathways and terminal distribution regions only expressed class I [28
]. As retinal grafts did not undergo rejection when placed in brains up to 8 days of age, we explored how this correlated with MHC expression in the developing optic pathway and with the onset of myelination. It was found that myelination began at 8–11 days of age; before this, optic nerve injury induced only MHC class I expression, and class II expression appeared at 10 days of age [29
]. The implication, therefore, was that grafts did not survive in a region in which host microglia were expressing class II MHC antigens and were therefore potentially antigen-presenting cells (APC). Products of degenerating myelin were effective in provoking class II MHC expression and subsequent graft rejection. Thus, suppression of MHC expression by microglia may be an important component of the immune protection afforded in this immunologically privileged site.
From this work, graft survival in immature brains may be indicative of a neonatal tolerance that can nevertheless be broken by subsequent inflammatory responses.
However, grafts placed in more mature brains were susceptible to rapid graft rejection. Although mismatched grafts could survive transplantation into a CNS site at an early age, they did not go unrecognized by the host immune system. Opening the BBB still led to rejection, as did specific immune challenge outside the CNS. Graft survival in immature brains may be indicative of a neonatal tolerance that can nevertheless be broken by a subsequent provocation.
In conclusion, up-regulation of host MHC class II molecules on microglia (thus enhancing their capacity to present alloantigen) appears to be a major factor contributing to rejection of otherwise stable grafts. Other factors that may contribute include the possibilities that grafted cells may migrate out of the transplant into the host tissue, and opening the BBB at the time of implantation or later might expose the "protected" graft to immune surveillance.
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PHOTORECEPTOR DEGENERATION AND INTRARETINAL GRAFTING
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Retinal degenerative diseases fall into two main groups: those affecting primarily the photoreceptors [involving a majority of cases of retinitis pigmentosa (RP) and related diseases] and those thought to involve the adjacent RPE, being responsible for a majority of cases of age-related macular degeneration (AMD) and a small number classified as RP. Of the first group, most are a result of single-gene mutations, with mutations in more than 100 genes so far identified contributing to photoreceptor death [30
]. Of the second group, although a relatively small number of specific mutations have been identified, many more cases are thought to be a result of polygenic causes, possibly with a strong environmental component. Although it is thought that the final phenotype of the disease may have many causes, a classification has yet to be achieved. Like the RP group of diseases, no suitable treatment has been found, although approaches, such as surgical intervention, and laser treatment regimens, such as photodynamic therapy, may slow the progress of specific consequences of the disease (see ref. [31
]).
The work on intracerebral retinal transplantation showed that functional relay circuits could be achieved after transplantation, and work ongoing at the same time on animal models of Parkinson’s disease (e.g., ref. [32
]) showed that grafts could also function in a more diffuse way, providing a missing chemical (in this case dopamine) to a region deprived of it. Such success led several groups to explore the important possibility of using transplantation as a potential therapy for neurodegenerative retinal diseases affecting the photoreceptors [33
34
35
36
37
38
39
40
41
]. The approach requires the implantation of cells into the subretinal space between the RPE layer and neural retina. In many of the animal studies, particularly those involving small animals, the space is usually accessed by introducing a pipette or other device through the sclera, choriocapillaris, and Bruch’s membrane; in human and larger animal studies, access is typically through the vitreous, entering the subretinal space through a retinotomy. This difference clearly has implications for potential immune responses, as any leakage of cells through a sclerotomy will most likely lead to peripheral sensitization to alloantigens and abrogate any immune privilege afforded the eye. Leakage into the vitreous may be less damaging, as it is a relatively confined space, and it appears to share certain immunosuppressive properties with aqueous humor, e.g., production of soluble Fas ligand (Fas-L) during ocular inflammation. There are two fundamentally different approaches to retinal transplantation. One is to limit the progress of photoreceptor loss by introducing cells before such loss has progressed too far, i.e., maintaining surviving photoreceptors. The other is to replace the photoreceptors once they have gone. For each, there are a number of common problems. These include choice of suitable cells for transplantation, method of transplantation (as dissociated cells or as a tissue), postoperative inflammatory or immune responses and how to minimize them, mode of transplant action, optimization of efficacy, and evaluation of success.
Maintenance of photoreceptors
To limit the progress of degeneration, the strategy used depends on the primary cause of the photoreceptor loss. For instance, if RPE cells are defective or lost, then replacement with "healthy" cells would be appropriate. Alternatively, the introduction of a cell type that "improves the chemical environment" of the subretinal space, perhaps by growth-factor release, could increase the efficiency of dysfunctional cells. In the former category, freshly harvested RPE cells have been used as donor cells. In earlier studies, these cells were grafted into the subretinal space of the Royal College of Surgeons (RCS) rat, which has a tyrosine receptor kinase (Mertk) defect affecting outer segment phagocytosis, leading to photoreceptor cell death [42
]. Grafts of fresh RPE cells have been shown to limit the progress of photoreceptor loss and preserve visual function [43
]. This same approach has been applied in clinical studies on patients with age-related macular degeneration, where the primary defect also lies in the RPE, albeit with a different origin [44
45
46
]. Although these studies have shown very little improvement in vision, this is not unexpected, as most patients had end-stage disease, where secondary degenerative changes in the inner retina probably had precluded functional improvement. Furthermore, unlike RPE cells derived from immature donors, RPE cells derived from adult donor tissue show poorer adhesion to Bruch’s membrane [47
]. As there was no attempt at tissue-matching in the human studies, there was also the possibility of graft rejection. Evidence for this was surmised from fluorescein angiography and fundus images [48
] but was not explored directly. In one case where triple immunosuppression was used [49
], and the patient died 114 days after surgery, retinal histology showed that clusters of round, pigmented cells survived, but the adjacent outer nuclear layer was disrupted. There was evidence of cystic spaces in the retina and lymphocytes in the choroid adjacent to the graft but no evidence of lymphocytes within it. Again, there was a lack of visual improvement after treatment. Apart from these concerns, there is a greater possibility of transmission of infection with fresh, harvested cells, as cultured cells could be carefully screened to reduce such risks. For these reasons as well as logistic and ethical considerations, fresh RPE cells do not seem ideal donor material for clinical allograft transplantation. An alternative approach is to obtain donor cells from another region of the affected eye. One group of studies has examined the use of autologous RPE grafts from a more peripheral part of the same eye [50
]. Others have explored the use of autologous grafts from the iris pigment epithelium (IPE), using animal models and humans with AMD [51
, 52
]. Work in animals has shown that IPE cells can be expanded in vitro and then grafted into the eye. The donor cells survive and can promote photoreceptor rescue [53
, 54
]. Human studies have also shown some indication of functional rescue [55
].
Immortalized RPE cells provide another source of donor material. Such cells implanted to the subretinal space of 3-week-old dystrophic RCS rats (Figs. 1
and 2
) can rescue a substantial photoreceptor layer over about one-quarter of the retinal quadrant, and this rescue can be achieved for periods exceeding 5 months [56
]. The rescued photoreceptors will generate an electroretinography (ERG) response with a prominent b-wave [57
], and when measuring response-sensitivity across the retina by recording multiunit activity in the superior colliculus to light flashes, the best responses differ very little from normal [58
].
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Figure 1. Diagrammatic representation of the transplantation procedure. Cells are grown in culture medium, trypsinized from the flask, and suspended in medium. The cell suspension is injected into the subretinal space using fine diameter glass capillary tubing attached to a 10-µl Hamilton syringe. Animals are assessed for visual function using head-tracking behavior and/or electrophysiology.
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Figure 2. (A) An unoperated rodent retina, viewed under the microscope through a dilated pupil. (B) The retinal detachment (*, opaque area) induced by the transplantation procedure. (Picture courtesy of A. S. L. Kwan.)
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Photoreceptor rescue has also been affected in various rodent models of retinal degeneration by improving the chemical environment by injecting growth factors directly into the eye or introducing them by adenoviral transfer (e.g., refs. [59
60
61
62
]). It should be noted, however, that there is a clear immune response to adenovirus injected into the anterior chamber or subretinal space [63
], which is an important factor in limiting the duration of adenovirally mediated gene expression in the subretinal space. This problem may be obviated by using a newer generation high-capacity adenovirus, which appears to be less immunogenic in subretinal injections [64
]. The other alternative is to deliver relevant growth factors using cells that naturally secrete them. One such cell is the Schwann cell, which makes a range of factors including brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor, ciliary neurotropic factor, and glial cell-derived neurotrophic factor (GDNF). When syngeneic Schwann cells are transplanted to the subretinal space of RCS rats, they survive for long periods, interspersed with RPE cells. In the region in which they are located, photoreceptor survival is prominent, and substantial outer segment maintenance can be seen. Behavioral studies measuring head-tracking responses to rotating vertical stripes and physiological studies measuring retinal sensitivity, recorded from the superior colliculus, indicate that there is good functional rescue using such cells [65
]. Anatomical rescue can also be seen in the rhodopsin knockout transgenic mouse [66
]. Recent work has shown that functional rescue can be enhanced in the dystrophic RCS rat using Schwann cell lines that have been transfected to produce GDNF or BDNF [67
].
One additional approach, designed specifically to limit the degeneration of cones, uses grafts containing high densities of rods [68
69
70
]. Rods have been shown to produce trophic factors, which support cone survival and after transplantation, limit the progress of cone degeneration [68
]. The identification of these rod-derived cone-survival factors and their molecular cloning should lead to future gene therapy and transplantation strategies.
Although cell transplantation to protect photoreceptors is largely studied with a view to alleviating or reversing the effects of AMD, the studies on transgenic mice suggest there may be a use in early onset diseases within the RP group. As the patients will be young, questions of donor cell safety and immune rejection become even more important.
Photoreceptor replacement
Photoreceptor replacement strategies have used dissociated or finely chopped pieces of immature retina [71
] or retinal sheets, [72
] intact or enriched in photoreceptors by vibratome or laser cutting. There is evidence from both approaches of some integration with the host. This is most successful in rabbit eyes [73
, 74
], somewhat less so in rats [75
76
77
], and poor in pigs [78
]. The favorable outcome in the rabbit studies may relate to the almost complete absence of an intrinsic retinal blood supply in this animal [79
] and a reliance on diffusion from the choriocapillaris to maintain the retina. In this transplant paradigm, the donor cells must establish a functional relationship with ancillary cells (RPE or Müller cells) involved in the photopigment cycling. They must also form synaptic connections with the remaining host retina, and these connections must be sufficiently organized to provide enough information for the visual centers of the brain to elaborate percepts and drive visual reflexes. Photoreceptor replacement strategies may be compromised, as loss of input from photoreceptors can lead to reorganization of synaptic connections or to the death of other retinal neurons. In turn, retinal cell loss can lead to synaptic remodeling in the central visual centers. Therefore, in this category of transplant, it is important that intervention occurs at an early time point in the degenerative process before regressive changes become too advanced. Again, the need for early intervention for optimal efficacy requires that questions of cell safety and tissue histocompatibility are resolved.
Use of stem cells
A further approach to transplantation, which is attracting great attention, is the use stem or progenitor cells. These cells could function by supporting or replacing photoreceptors. Although stem cells derived from the adult hippocampus have been shown to integrate with the host retina, adopting neuronal and glial profiles, they failed to express retinal markers [80
81
82
]. However, if treated before transplantation, there is some indication that cells will express retina-specific markers. Furthermore, retina-derived progenitor cells will differentiate into a range of retinal cell types after isolation in vitro [83
]. The possibility that embryonic stem cells may also serve to provide a source of photoreceptors is being explored and shows promise. Although immune responses to adult and embryonic stem cells are minimal, the up-regulation of MHC, CD45, and CD11 markers on transplanted cells and their neighbors might still result in graft rejection [84
].
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TRANSPLANT FUNCTION AND MAINTENANCE OF VISION
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There are two principal questions: First, how well can various substrates of vision be restored or maintained by transplantation, and second, how do the grafts actually work?
In relation to the first, very little attention was devoted to the question of function until quite recently. For grafts designed to limit the course of degeneration, a range of behavioral tests has now been used, including pupillary light reflex [9
, 85
], head-tracking to moving stripes (Fig. 3
) [86
], and discrimination of stripes of different spatial frequencies [56
, 87
].
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Figure 3. Head-tracking apparatus. The rat is placed in a perspex cylinder. A drum rotates around this, clockwise or counter-clockwise, displaying striped gratings of various periodicities (0.5, 0.25, 0.125 cycles per degree) to the rat. A sighted animal will follow the moving stripes, and the time spent tracking the movement can be recorded.
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Physiological studies examining ERG, light adaptation curves, threshold responses recorded from the superior colliculus (Fig. 4
) [57
, 88
], and single-unit responses recorded from the visual cortex have also been used [56
].
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Figure 4. Diagrammatic representation of electrophysiological recording of visual function. (A) Responses to a light stimulus (log candela/m2; presented across a Ganzfield bowl) are recorded from the superior colliculus. (B) Multiunit responses to light flash are recorded and thresholds determined. (C) By recording from 76 separate points across the colliculus, it is possible to generate a threshold-sensitivity map. Areas with the lowest response-threshold correspond to regions of photoreceptors rescued by the transplant.
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This work has all been done in rodents. The results have shown improved performance compared with controls on amplitude of the pupillary light reflex. However, the recent studies show there are unexpectedly high background levels, which may be a result of the fact that a response can also be elicited through melanopsin-containing ganglion cells [89
, 90
]. Head-tracking gives an effective and simple indicator of transplant efficacy, which at high spatial resolutions (0.5 cycles/degree), requires an intact visual cortex. Visual discrimination tests show how well an animal can "see" and specifically have been used for studying acuity performance; again, protective cell grafts can preserve some level of function. ERG responses may also be preserved, although it is clear that this test is not particularly sensitive. A recent systematic study [57
] has shown that a b-wave can be preserved for many months in RCS rats with RPE cell-line transplants, and a b-wave can no longer be detected when the threshold sensitivity response is worse than 2.5 log candela/m2 over more than 50% of the visual field. Threshold responses to a spot of light recorded across the area of the superior colliculus give an effective measure of retinal sensitivity across the visual field. This has shown that sustained rescue of low-sensitivity responses in an area of grafting can be maintained for many months post-transplantation. Finally, unit responses recorded in the visual cortex show rescue of elaborate and detailed tuning of single cells to presentation of particular stimulus parameters.
Studies on the functional sequelae of transplants designed to reconstruct retinal circuitry are very much less complete. There is some indication of restoration of photophobic responses [71
], of low threshold responses in the superior colliculus to spot or full-field light flashes [91
], and of some restoration of ganglion cell activity after transplantation [92
].
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INTERPRETATION OF FUNCTIONAL STUDIES
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The use of function as an outcome of efficacy is an important step in this field, but how these various grafts work to produce improved performance better than controls is presently an enigma. It is assumed that in the case of grafted RPE cells or photoreceptors, they take on the function of the cells that they replace, but this has been surmised without direct proof. For RPE cells, there is a paucity of work identifying donor cells, and none of this has shown that they are capable of phagocytosing outer segments at normal rates or of performing other essential functions of RPE cells. Possibly, they might induce host cells to function more effectively. Whatever the mechanism, studies using RPE cell lines transplanted to RCS rats show that a variety of functions, including conscious vision, can be maintained at levels that lie somewhere between normal performance and sham-operated animals. Studies using Schwann cells have also indicated improved retinal sensitivity over controls [65
]. Function has not been investigated using other donor cell types in experimental animals, although the functional consequences of using IPE cells in humans have been evaluated.
In studies investigating photoreceptor replacement, it is apparent that such grafts do form a synaptic interface with host retina [71
, 93
], but it is unclear whether this is sufficient to relay useful information to the CNS to affect visual perception. Whether the functional improvement is a result of reconstructed connections or simply improvement in remaining cone performance is unclear at this point.
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IMMUNE REJECTION
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The immune status of the subretinal space is atypical [94
, 95
]. Gross infiltration by lymphocytes is rarely seen after allografting, and the subretinal space has been considered to be a partially immunologically privileged site. It exhibits a phenomenon referred to as anterior chamber-associated immune deviation (ACAID) [96
97
98
]. This response to foreign antigens is an active, systemic process leading to down-regulation of the normal delayed-type hypersensitivity (DTH) response seen after transplantation of tissue to nonimmune-privileged sites [99
, 100
]. Locally produced cytokines {e.g., transforming growth factor-ß (TGF-ß) and interleukin (IL)-10 [101
, 102
]} and the generation of regulatory T cells in the spleen are thought to alter the immune response from the T helper cell type 1 (Th1) population seen in DTH to the Th2 type [103
104
105
]. TGF-ß2 activates thrombospondin gene expression in ocular APC and inhibits IL-12 and CD40 expression [106
]. Lymphocytes derived from the spleens of these animals are capable of suppressing DTH when adoptively transferred to naïve individuals [107
]. Studies have shown that this process requires an intact blood-retinal barrier (BRB) [107
].
Although prolonged graft survival has been described [108
, 109
], foreign tissue in the subretinal space is not necessarily protected from immune attack [107
, 110
111
112
], and allogeneic grafts can still be lost despite immunosuppressive therapy (cyclosporine A [113
]), indicating possible non-T cell-dependent graft destruction. Nonimmune-mediated destruction of grafted cells must be considered, such as Fas (CD-95) and Fas-L (CD-95 L) interactions. This interaction leads to the apoptosis of Fas-expressing, activated T cells [114
, 115
]. RPE cells express Fas-L [115
116
117
], and this may be important in down-regulating activated T cells in the vicinity [118
]. RPE cells also constitutively express Fas but are resistant to Fas/Fas-L-dependent apoptosis under normal conditions. However, in inflammatory conditions such as proliferative vitreo-retinopathy, this protection is lost (possibly by inhibition of RNA or protein synthesis), and these cells can be apoptosed via the Fas/Fas-L pathway [116
]. This may be one mechanism contributing to graft cell loss post-transplantation.
Mismatch of MHC haplotypes (especially class I; ref. [118
]) can lead to graft rejection, as does the up-regulation of MHC class II expression in graft cells [119
]. Antigen must be presented to host T cells in association with MHC II molecules [120
] to provoke graft rejection, and there is some up-regulation of MHC II expression in subretinal grafts [109
, 121
]. The identity of the APC in the donor neural retina is not clear, but microglia (termed passenger leukocytes by Ma and Streilein [122
]) are likely candidates, and the observed increase in MHC II expression may be on these microglia, as well as on donor RPE cells. The importance of the APC in the process of graft rejection in the CNS makes the presentation of alloantigen via the indirect pathway very likely.
Any immune privilege is likely to be compromised if the BRB is disrupted, as this will expose the retina to immune surveillance [107
, 123
]. As vascular integrity is lost in some human retinal diseases, notably RP and neovascular AMD, and some animal models (e.g., the dystrophic RCS rat [124
] and the rd mouse [125
]), potential problems of transplantation into the subretinal space with a persistently compromised BRB cannot be ignored.
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FUTURE DEVELOPMENTS TO IMPROVE GRAFT EFFICACY
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Improvement of donor cell environment
In other CNS transplant paradigms, it has been found that a large proportion of transplanted neurons die within 1 week of transplantation, by necrosis or by apoptosis (see review by Boonman and Isacson [126
]). Some of this loss may be a result of damage caused by the preparation of the donor cell suspension [127
] or to the transplantation process, but lack of oxygen or appropriate growth factors could also be involved. Accordingly, lazaroids (21 aminosteroids [128
129
130
131
]), growth factors [132
, 133
], and inhibitors of apoptosis [134
] have been used to improve the chemical environment of the cells before and after grafting. Although direct evidence for such cell losses is lacking in the retina, the use of specific maintenance factors such as neurotrophins or lazaroids should be explored.
Improvements for RPE cell transplantation
There are a number of problems specific to RPE cell transplantation. Crucially, RPE cells must attach to Bruch’s membrane. Any impediment to grafted RPE cells accessing Bruch’s membrane, such as host RPE cells, would prejudice against graft survival and lead to anchorage-dependent apoptosis (anoikis). Furthermore, it has been shown that a damaged Bruch’s membrane is much less effective as a substrate for RPE cell attachment (e.g., ref. [135
]). Chemical manipulation of Bruch’s membrane or of the donor cells before transplantation might improve adhesion and with it, cell survival. Improvements to short-term cell survival will be crucial if questions relating to the delayed graft loss mechanism seen in allograft rejection are to be investigated.
Improvements to photoreceptor-replacement strategies
For grafts intended to replace lost photoreceptors, it is first necessary to circumvent the host glial reactions that accompany photoreceptor loss. Second, the grafts must make sensible connections with the remaining cells of the host retina. It is clear that in the course of retinal degeneration, physical or chemical barriers are created in the retina and in the CNS, which may prevent reconstruction of neural pathways. In other studies, e.g., spinal cord repair, improved regeneration can be achieved by modulating these barriers [136
, 137
]. Whether similar approaches are necessary in the retina has yet to be explored.
Value of training animals in visual tasks
Functional efficacy might be improved by training the system to work. There is a large literature in other degeneration conditions and after transplantation, showing that training can sometimes optimize efficacy (e.g., refs. [11
, 138
]). This has not been explored for intraretinal grafts.
Minimizing postoperative inflammatory or immune responses
Many questions remain unanswered in this area. For instance: (i) What are the substrates of immune privilege in the subretinal space? Can a "classical" immune response ever be evoked in the retina, for instance, by selection of donor/host rodent strains known to invoke a rejection response in the brain, also originally believed to be an immune-privileged site? (ii) If graft rejection does occur, what is the mechanism involved? (iii) What is the effect of the transplantation process on an immune-privileged site? Is immune privilege in the subretinal space retained during intraocular inflammation as occurs in ACAID? Does the opening of the BRB, which necessarily occurs at the time of grafting, expose the graft to immune surveillance, and how can allorecognition be prevented? Different types of immune suppression need to be assessed and the time course of their application evaluated. For example, it may only be necessary to suppress the host-immune system while the BRB is open. Donor cells could be manipulated to reduce host-tissue responses. Additional strategies to tolerate recipients to RPE allografts also need to be tested. (iv) As grafts can rarely protect the whole retina from degeneration, what will be the effect of development of the "leaky" vessels that often accompany outer retinal degeneration in rodents and humans? Will mismatch graft survival then be compromised? (v) Do the reactive responses to photoreceptor degeneration affect the afferent limb of immune recognition? These include microglial and macrophage invasion into the outer retina and potential up-regulation of antigen-presenting capability.
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CONCLUSION
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The work, summarized very briefly above and discussed in greater detail in the reviews and papers referenced, shows that cells can survive transplantation to the diseased eye in experimental animals, maintaining or restoring some degree of function. The extrapolation of these successes to humans is still to be demonstrated definitively, but progress is being made to this end. Whether graft survival can be enhanced by other treatments or by attention to immune considerations remains to be seen. Work on transplantation to the CNS has shown that even stable, mismatched grafts are susceptible to rejection under specific conditions. How that work relates to intraretinal grafting has yet to be explored, especially as substantial T cell infiltration has yet to be reported after retinal grafting. Clearly, a greater knowledge of the immune and inflammatory components that accompany the degenerative events and the introduction of specific transplants represents a major area for further investigation.
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ACKNOWLEDGEMENTS
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The personal studies summarized here have been supported by grants from NIH, Wellcome Trust, MRC (UK), Foundation Fighting Blindness, and Research to Prevent Blindness. R. D. L. is the Calvin and JeNeal Hatch Professor of Ophthalmology at the University of Utah. S. J. O. is the Glaxo-Smith Kline Professor of Biomedical Science at the Institute of Ophthalmology, UCL. We thank all our collaborators, who over the years have contributed to various parts of the work that have led to this synthesis.
Received January 24, 2003;
revised April 30, 2003;
accepted May 2, 2003.
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ABSTRACT
INTRODUCTION